This invention relates to iodinated and radioiodinated compounds which are useful as biological reagents and tracers, and more specifically, to iodinated and radioiodinated diacylglycerol analogues.
Diacylglycerols have recently been attracting attention because of their ability to activate protein kinase C (hereinafter PK-C). PK-C is a phosphorylating enzyme which is believed to be involved in signal transduction and tumor promotion by phorbol diesters and related compounds. Phospholipid and calcium are required for activation of the PK-C enzyme. The affinity of PK-C for Ca.sup.+2 ions is increased by diacylglycerol, which is transiently produced by the action of phospholipase C on phosphatidylinositol 4,5-bisphosphate in response to biological stimuli. The binding of diacylglycerols to PK-C is stoichiometric and competitive with phorbol diesters. Both diacylglycerols and phorbol diesters activate the PK-C enzyme, which, once activated, phosphorylates any of a number of proteins such as histone H1, myelin basic protein, cardiac troponin I and T, and a 40 kilodalton protein in platelets. In this way, diacylglycerol acts as a second messenger in the production of a biological response.
It appears that diacylglycerols and phorbol diesters have differences in their mechanisms of action. A possible explanation for this is that phorbol diesters are not metabolized as readily as diacylglycerols. It is known that phorbol diesters are not substrates for diacylglycerol lipase or diacylglycerol kinase, the enzymes which are primarily responsible for inactivation of diacylglycerols. Elucidation of the differences between these two types of compounds may lead to a greater understanding of the mechanism of tumor promotion by phorbol diesters. Therefore, a biological probe for identification and characterization of PK-C binding sites and mechanisms of action of diacylglycerols would be of great significance to investigators in this field. Moreover, such a probe would enable researchers to ascertain whether synthesized diacylglycerol analogues could be modifiers of the normal biological action of natural occurring diacylglycerols, and thus could have pharmacological effect.
Currently, tritiated phorbol diesters, such as [.sup.3 H]phorbol dibutyrate, are available for use in studying the mechanism of action of phorbol diesters in tumor promotion. There are currently no radioactive diacylglycerol analogues available for studying the mechanism of action of diacylglycerols. While tritiated compounds are radioemissive, the emission is strictly of the week beta-type. Thus, any interfering structure, such as nearby tissue in vivo, completely absorbs or at least substantially masks the emission before the .sup.3 H can be detected. Therefore, beta-measuring instruments must be highly sensitive and intervening tissue absorption must be factored-in, thereby increasing the problems created by the use of beta-emissive radio-tagged compounds in laboratory situations. There is therefore a need for radio-tagged compounds for binding to PK-C sites having relatively strong emissive characteristics, as such would be of substantial benefit to investigators of such reactions at the molecular level.
Moreover, PK-C is present in high concentrations in brain tissue and certain tumors, such as leukemia tumors. Since diacylglycerols bind to PK-C, a radioactive form of diacylglycerols would be useful as a non-invasive site-specific imaging agent for organs and tumors where PK-C is concentrated.
Two of the most widely used imaging modalities, radiography and radioisotope scanning, owe much of their success to the development of suitable radiopaques and radiopharmaceuticals. Iodine has played an important role in the development of suitable radiopaque and radiopharmaceutical compounds. Iodine not only imparts the necessary electron density to radiopaques; but can, in its various isotope states (for example, the clinically used isotopes: I.sup.123, I.sup.125, and I.sup.131), emit gamma radiation essential for gamma-camera scintigraphy. Thus, there is a need for iodinated and/or radioiodinated diacylglycerol analogues which are physiologically acceptable and non-toxic for administration to a living being for noninvasive imaging techniques. There is additionally a need for radioactive forms of diacylglycerols for use in tissue distribution studies, and as probes in determining uptake of diacylglycerols into intact cells, in cellular distribution studies, translocation of PK-C from cytosol to membrane upon activation, and biological responses to PK-C activation.
It is, therefore, an object of this invention to provide novel analogues of diacylglycerol, in particular, novel iodinated and radioiodinated analogues of diacylglycerol.
It is another object of this invention to provide novel iodinated and radioiodinated analogues of diacylglycerol as modifiers of PK-C function.
It is also an object of this invention to provide novel iodinated and radioiodinated analogues of diacylglycerol as radiopaques and radiopharmaceuticals.
It is a further object of this invention to provide novel iodinated and radioiodinated analogues of diacylglycerol as radiopaques and radiopharmaceuticals which are physiologically acceptable and non-toxic.
It is additionally an object of this invention to provide novel iodinated analogues of diacylglycerol which will opacify soft tissue when administered in small doses.
It is yet a further object of this invention to provide novel iodinated analogues of diacylglycerol which can be radiotagged for use in scintigraphy.
It is also another object of this invention to provide novel radioiodinated analogues of diacylglycerol which will be cleared from the body of a living being to which it has been administered within a reasonable period of time, while also being sufficiently stable to permit adequate residence time for high-quality imaging.
It is yet an additional object of this invention to provide novel iodinated and radioiodinated diacylglycerol analogues which can be prepared and analyzed more easily than currently available tritiated phorbol diester compounds.
It is still another object of this invention to provide novel iodinated and radioiodinated diacylglycerol analogues which have a higher specific activity than currently available tritiated phorbol diester compounds.
It is a yet further object of this invention to provide novel techniques and methods of making iodinated and radioiodinated diacylglycerol analogues.
It is also a further object of this invention to provide iodinated and radioiodinated diacylglycerol analogues as tracers for monitoring the interaction of such analogues with PK-C both in vitro and in vivo for characterizing the binding site and its role in cell proliferation.